RNA-Seq: differential expression analysis of controls
Libraries required
Filtering gene counts
Function
Differeitial analysis using limma
SVA
se <- SummarizedExperiment(assays = counts.filt)
se@colData <- DataFrame(salmon@phenoData)
sv.pl <- svacor(SE = se, form = ~ 0 + Group)## Using variance-stabilizing transformation
## converting counts to integer mode
## Warning in DESeqDataSet(se, design = design, ignoreRank): some variables in
## design formula are characters, converting to factors
## Number of significant surrogate variables is: 4
## Iteration (out of 5 ):1 2 3 4 5
limma fit
design <- model.matrix(~ 0 + SV1 + SV2 + SV3 + Group, data = sv.pl@colData)
colnames(design) <- gsub(pattern = "Group", replacement = "", x = colnames(design))
dds <- calcNormFactors(DGEList(counts = counts.filt))
v <- voom(dds, design = design)
contrast.matrix <- makeContrasts(PND15 - PND8, Adult - PND15, levels = design)
fit <- lmFit(v)
fit2 <- contrasts.fit(fit, contrast.matrix)
fit2 <- eBayes(fit2)DEA results
pnd8.pnd15 <- as.data.frame(topTable(fit2, coef = 1, number = Inf))
pnd8.pnd15 <- data.frame(Genes = rownames(pnd8.pnd15), pnd8.pnd15, stringsAsFactors = F)
pnd15.adult <- as.data.frame(topTable(fit2, coef = 2, number = Inf))
pnd15.adult <- data.frame(Genes = rownames(pnd15.adult), pnd15.adult, stringsAsFactors = F)vst Counts data and pData
dge <- DGEList(counts = counts.filt, group = salmon@phenoData$Group)
dge <- calcNormFactors(object = dge, method = "TMM")
cpm <- cpm(dge, log = T)
cpm <- removeBatchEffect(x = cpm, covariates = design[, 1:3], design = design[, 4:6])
data <- list(cpm = cpm, vstSV = assay(sv.pl), voomE = v$E, pData = salmon@phenoData)
save(data,
file = "./output/data_pData_tx.RData", compress = T,
compression_level = 3
)Results
dea.list <- list(
`PND8 vs PND15` = as.DEA(pnd8.pnd15),
`PND15 vs Adult` = as.DEA(pnd15.adult)
)
normCounts <- v$E
voomEList <- v
dea.limma <- list(
`PND8 vs PND15` = pnd8.pnd15,
`PND15 vs Adult` = pnd15.adult
)Save RData files
save(dea.list,
file = "./output/tx_dea_SC_Controls.DEA.RData", compress = T,
compression_level = 3
)
save(normCounts,
file = "./output/tx_normCounts_voom_SC_Controls.RData", compress = T,
compression_level = 3
)
save(voomEList,
file = "./output/tx_voom_EList_SC_Controls.RData", compress = T,
compression_level = 3
)
save(dea.limma,
file = "./output/tx_limma_SC_Controls.RData", compress = T,
compression_level = 3
)PCA plots
Raw counts filtered
SVA (VST)
SessionInfo
─ Session info ───────────────────────────────────────────────────────────────
setting value
version R version 3.6.2 (2019-12-12)
os Ubuntu 16.04.6 LTS
system x86_64, linux-gnu
ui X11
language (EN)
collate en_US.UTF-8
ctype en_US.UTF-8
tz Europe/Zurich
date 2020-07-08
─ Packages ───────────────────────────────────────────────────────────────────
package * version date lib
acepack 1.4.1 2016-10-29 [1]
annotate 1.64.0 2019-10-29 [1]
AnnotationDbi 1.48.0 2019-10-29 [1]
assertthat 0.2.1 2019-03-21 [1]
backports 1.1.7 2020-05-13 [1]
base64enc 0.1-3 2015-07-28 [1]
Biobase * 2.46.0 2019-10-29 [1]
BiocGenerics * 0.32.0 2019-10-29 [1]
BiocParallel * 1.20.1 2019-12-21 [1]
Biostrings 2.54.0 2019-10-29 [1]
bit 1.1-15.2 2020-02-10 [1]
bit64 0.9-7 2017-05-08 [1]
bitops 1.0-6 2013-08-17 [1]
blob 1.2.1 2020-01-20 [1]
bookdown 0.18 2020-03-05 [1]
callr 3.4.3 2020-03-28 [1]
checkmate 2.0.0 2020-02-06 [1]
cli 2.0.2 2020-02-28 [1]
cluster 2.1.0 2019-06-19 [1]
colorspace 1.4-1 2019-03-18 [1]
crayon 1.3.4 2017-09-16 [1]
crosstalk 1.1.0.1 2020-03-13 [1]
data.table 1.12.8 2019-12-09 [1]
DBI 1.1.0 2019-12-15 [1]
DelayedArray * 0.12.3 2020-04-09 [1]
desc 1.2.0 2018-05-01 [1]
DESeq2 * 1.26.0 2019-10-29 [1]
devtools 2.3.0 2020-04-10 [1]
digest 0.6.25 2020-02-23 [1]
dplyr * 1.0.0 2020-05-29 [1]
edgeR * 3.28.1 2020-02-26 [1]
ellipsis 0.3.1 2020-05-15 [1]
evaluate 0.14 2019-05-28 [1]
fansi 0.4.1 2020-01-08 [1]
farver 2.0.3 2020-01-16 [1]
foreign 0.8-76 2020-03-03 [1]
Formula 1.2-3 2018-05-03 [1]
fs 1.4.1 2020-04-04 [1]
genefilter * 1.68.0 2019-10-29 [1]
geneplotter 1.64.0 2019-10-29 [1]
generics 0.0.2 2018-11-29 [1]
GenomeInfoDb * 1.22.1 2020-03-27 [1]
GenomeInfoDbData 1.2.2 2019-11-18 [1]
GenomicRanges * 1.38.0 2019-10-29 [1]
GEOquery 2.54.1 2019-11-18 [1]
ggplot2 * 3.3.1 2020-05-28 [1]
glue 1.4.1 2020-05-13 [1]
gridExtra 2.3 2017-09-09 [1]
gtable 0.3.0 2019-03-25 [1]
Hmisc 4.4-0 2020-03-23 [1]
hms 0.5.3 2020-01-08 [1]
htmlTable 1.13.3 2019-12-04 [1]
htmltools 0.4.0 2019-10-04 [1]
htmlwidgets 1.5.1 2019-10-08 [1]
httr 1.4.1 2019-08-05 [1]
IRanges * 2.20.2 2020-01-13 [1]
jpeg 0.1-8.1 2019-10-24 [1]
jsonlite 1.6.1 2020-02-02 [1]
knitr 1.28 2020-02-06 [1]
lattice 0.20-41 2020-04-02 [1]
latticeExtra 0.6-29 2019-12-19 [1]
lazyeval 0.2.2 2019-03-15 [1]
lifecycle 0.2.0 2020-03-06 [1]
limma * 3.42.2 2020-02-03 [1]
locfit 1.5-9.4 2020-03-25 [1]
magrittr 1.5 2014-11-22 [1]
Matrix 1.2-18 2019-11-27 [1]
matrixStats * 0.56.0 2020-03-13 [1]
memoise 1.1.0.9000 2020-05-06 [1]
mgcv * 1.8-31 2019-11-09 [1]
munsell 0.5.0 2018-06-12 [1]
nlme * 3.1-147 2020-04-13 [1]
nnet 7.3-13 2020-02-25 [1]
pillar 1.4.4 2020-05-05 [1]
pkgbuild 1.0.8 2020-05-07 [1]
pkgconfig 2.0.3 2019-09-22 [1]
pkgload 1.1.0 2020-05-29 [1]
plgINS * 0.1.5 2020-07-08 [1]
plotly * 4.9.2.1 2020-04-04 [1]
plyr 1.8.6 2020-03-03 [1]
png 0.1-7 2013-12-03 [1]
prettyunits 1.1.1 2020-01-24 [1]
processx 3.4.2 2020-02-09 [1]
ps 1.3.3 2020-05-08 [1]
purrr 0.3.4 2020-04-17 [1]
R6 2.4.1 2019-11-12 [1]
RColorBrewer 1.1-2 2014-12-07 [1]
Rcpp 1.0.4.6 2020-04-09 [1]
RCurl 1.98-1.2 2020-04-18 [1]
readr 1.3.1 2018-12-21 [1]
remotes 2.1.1 2020-02-15 [1]
rlang 0.4.6 2020-05-02 [1]
rmarkdown 2.1 2020-01-20 [1]
rmdformats 0.4.0 2020-06-07 [1]
rpart 4.1-15 2019-04-12 [1]
rprojroot 1.3-2 2018-01-03 [1]
RSQLite 2.1.4 2019-12-04 [1]
rstudioapi 0.11 2020-02-07 [1]
S4Vectors * 0.24.4 2020-04-09 [1]
scales 1.1.1 2020-05-11 [1]
sessioninfo 1.1.1 2018-11-05 [1]
SRAdb 1.48.2 2019-12-24 [1]
stringi 1.4.6 2020-02-17 [1]
stringr 1.4.0 2019-02-10 [1]
SummarizedExperiment * 1.16.1 2019-12-19 [1]
survival 3.1-12 2020-04-10 [1]
sva * 3.34.0 2019-10-29 [1]
testthat 2.3.2 2020-03-02 [1]
tibble 3.0.1 2020-04-20 [1]
tidyr 1.1.0 2020-05-20 [1]
tidyselect 1.1.0 2020-05-11 [1]
usethis 1.6.1 2020-04-29 [1]
vctrs 0.3.1 2020-06-05 [1]
viridisLite 0.3.0 2018-02-01 [1]
withr 2.2.0 2020-04-20 [1]
xfun 0.13 2020-04-13 [1]
XML 3.99-0.3 2020-01-20 [1]
xml2 1.3.2 2020-04-23 [1]
xtable 1.8-4 2019-04-21 [1]
XVector 0.26.0 2019-10-29 [1]
yaml 2.2.1 2020-02-01 [1]
zlibbioc 1.32.0 2019-10-29 [1]
source
CRAN (R 3.6.1)
Bioconductor
Bioconductor
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
Bioconductor
Bioconductor
Bioconductor
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
Bioconductor
CRAN (R 3.6.1)
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
Bioconductor
Bioconductor
CRAN (R 3.6.1)
Bioconductor
Bioconductor
Bioconductor
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
Bioconductor
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
Github (r-lib/memoise@4aefd9f)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
local
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
Github (juba/rmdformats@94cd7a3)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.1)
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.1)
Bioconductor
CRAN (R 3.6.2)
Bioconductor
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.2)
CRAN (R 3.6.1)
Bioconductor
CRAN (R 3.6.2)
Bioconductor
[1] /home/ubuntu/R/x86_64-pc-linux-gnu-library/3.6
[2] /usr/local/lib/R/site-library
[3] /usr/lib/R/site-library
[4] /usr/lib/R/library